Signaling through the interleukin-4 and interleukin-13 receptor complexes regulates cholangiocyte TMEM16A expression and biliary secretion.

TMEM16A is a Ca2+-activated Cl- channel in the apical membrane of biliary epithelial cells, known as cholangiocytes, which contributes importantly to ductular bile formation. Whereas cholangiocyte TMEM16A activity is regulated by extracellular ATP-binding membrane purinergic receptors, channel expression is regulated by interleukin-4 (IL-4) through an unknown mechanism. Therefore, the aim of the present study was to identify the signaling pathways involved in TMEM16A expression and cholangiocyte secretion. Studies were performed in polarized normal rat cholangiocyte monolayers, human Mz-Cha-1 biliary cells, and cholangiocytes isolated from murine liver tissue. The results demonstrate that all the biliary models expressed the IL-4Rα/IL-13Rα1 receptor complex. Incubation of cholangiocytes with either IL-13 or IL-4 increased the expression of TMEM16A protein, which was associated with an increase in the magnitude of Ca2+-activated Cl- currents in response to ATP in single cells and the short-circuit current response in polarized monolayers. The IL-4- and IL-13-mediated increase in TMEM16A expression was also associated with an increase in STAT6 phosphorylation. Specific inhibition of JAK-3 inhibited the increase in TMEM16A expression and the IL-4-mediated increase in ATP-stimulated currents, whereas inhibition of STAT6 inhibited both IL-4- and IL-13-mediated increases in TMEM16A expression and ATP-stimulated secretion. These studies demonstrate that the cytokines IL-13 and IL-4 regulate the expression and function of biliary TMEM16A channels through a signaling pathway involving STAT6. Identification of this regulatory pathway provides new insight into biliary secretion and suggests new targets to enhance bile formation in the treatment of cholestatic liver disorders.NEW & NOTEWORTHY The Ca2+-activated Cl- channel transmembrane member 16A (TMEM16A) has emerged as an important regulator of biliary secretion and hence, ductular bile formation. The present studies represent the initial description of the regulation of TMEM16A expression in biliary epithelium. Identification of this regulatory pathway involving the IL-4 and IL-13 receptor complex and JAK-3 and STAT-6 signaling provides new insight into biliary secretion and suggests new therapeutic targets to enhance bile formation in the treatment of cholestatic liver disorders.

Mechanosensor transient receptor potential vanilloid member 4 (TRPV4) regulates mouse cholangiocyte secretion and bile formation.

Mechanosensitive signaling has emerged as a mechanism for the regulation of cholangiocyte transport and bile formation. The mechanical effect of fluid-flow, or shear, at the apical membrane of cholangiocytes regulates secretion through a process involving increases in [Ca2+]i and activation of Ca2+-activated Cl- channels. However, the initiating steps translating shear force to increases in intracellular calcium concentration ([Ca2+]i) are unknown. Transient receptor potential vanilloid member 4 (TRPV4), a nonselective cation channel present in the apical membrane of cholangiocytes, has been proposed as a potential mechanosensor. The aim of the present studies was to determine the potential role of TRPV4 in initiating mechanosensitive signaling in response to fluid-flow in cholangiocytes. TRPV4 expression was confirmed in both small and large mouse cholangiocytes. Exposure of cells to either fluid flow or specific TRPV4 pharmacological agonists rapidly increased both [Ca2+]i and membrane cation currents. Both flow- and agonist-stimulated currents displayed identical biophysical properties and were inhibited in the presence of TRPV4 antagonists or in cells after transfection with TRPV4 small interfering RNA. Transfection of mouse cholangiocytes with a TRPV4-enhanced green fluorescent protein construct increased the expression of TRPV4 and the magnitude of flow-stimulated currents. A specific TRPV4 agonist significantly increased the biliary concentration of ATP and bile flow in live mice when administered intravenously and increased ATP release from cholangiocyte monolayers when applied exogenously. The findings are consistent with a model in which activation of cholangiocyte TRPV4 translates shear force into an acute rise in membrane cation permeability, [Ca2+]i, ATP release, and bile flow. Understanding the role of mechanosensitive transport pathways may provide novel insights to modulate bile flow for the treatment of cholestatic liver disorders.NEW & NOTEWORTHY These studies functionally characterize TRPV4 as a mechanosensitive channel in mouse cholangiocytes. By mediating a rapid rise in intracellular Ca2+, necessary for Ca2+-dependent secretion, TRPV4 represents a mechanosensor responsible for translating fluid flow into intracellular signaling and biliary secretion. Furthermore, intravenous infusion of a specific TRPV4 agonist increases bile flow in live mice. Understanding the role of TRPV4 in mechanosensitive transport pathways may provide novel insights to modulate bile flow during cholestasis.

Bile acids stimulate cholangiocyte fluid secretion by activation of transmembrane member 16A Cl- channels.

Bile acids stimulate a bicarbonate-rich choleresis, in part, through effects on cholangiocytes. Because Cl- channels in the apical membrane of cholangiocytes provide the driving force for secretion and transmembrane member 16A (TMEM16A) has been identified as the Ca2+ -activated Cl- channel in the apical membrane of cholangiocytes, the aim of the present study was to determine whether TMEM16A is the target of bile-acid-stimulated Cl- secretion and to identify the regulatory pathway involved. In these studies of mouse, rat, and human biliary epithelium exposure to ursodeoxycholic acid (UDCA) or tauroursodeoxycholic acid (TUDCA) rapidly increased the rate of exocytosis, ATP release, [Ca2+ ]i , membrane Cl- permeability, and transepithelial secretion. Bile-acid-stimulated Cl- currents demonstrated biophysical properties consistent with TMEM16A and were inhibited by pharmacological or molecular (small-interfering RNA; siRNA) inhibition of TMEM16A. Bile acid-stimulated Cl- currents were not observed in the presence of apyrase, suramin, or 2-aminoethoxydiphenyl borate (2-APB), demonstrating that current activation requires extracellular ATP, P2Y, and inositol 1,4,5-trisphosphate (IP3) receptors. TUDCA did not activate Cl- currents during pharmacologic inhibition of the apical Na+ -dependent bile acid transporter (ASBT), but direct intracellular delivery of TUDCA rapidly activated Cl- currents. CONCLUSION: Bile acids stimulate Cl- secretion in mouse and human biliary cells through activation of membrane TMEM16A channels in a process regulated by extracellular ATP and [Ca2+ ]i . These studies suggest that TMEM16A channels may be targets to increase bile flow during cholestasis. (Hepatology 2018;68:187-199).

PKCα regulates TMEM16A-mediated Cl⁻ secretion in human biliary cells.

TMEM16A is a newly identified Ca(2+)-activated Cl(-) channel in biliary epithelial cells (BECs) that is important in biliary secretion. While extracellular ATP stimulates TMEM16A via binding P2 receptors and increasing intracellular Ca(2+) concentration ([Ca(2+)]i), the regulatory pathways have not been elucidated. Protein kinase C (PKC) contributes to ATP-mediated secretion in BECs, although its potential role in TMEM16A regulation is unknown. To determine whether PKCα regulates the TMEM16A-dependent membrane Cl(-) transport in BECs, studies were performed in human biliary Mz-cha-1 cells. Addition of extracellular ATP induced a rapid translocation of PKCα from the cytosol to the plasma membrane and activation of whole cell Ca(2+)-activated Cl(-) currents. Currents demonstrated outward rectification and reversal at 0 mV (properties consistent with TMEM16A) and were inhibited by either molecular (siRNA) or pharmacologic (PMA or Gö6976) inhibition of PKCα. Intracellular dialysis with recombinant PKCα activated Cl(-) currents with biophysical properties identical to TMEM16A in control cells but not in cells after transfection with TMEM16A siRNA. In conclusion, our studies demonstrate that PKCα is coupled to ATP-stimulated TMEM16A activation in BECs. Targeting this ATP-Ca(2+)-PKCα signaling pathway may represent a therapeutic strategy to increase biliary secretion and promote bile formation.

Regulation of mechanosensitive biliary epithelial transport by the epithelial Na(+) channel.

UNLABELLED: Intrahepatic biliary epithelial cells (BECs), also known as cholangiocytes, modulate the volume and composition of bile through the regulation of secretion and absorption. While mechanosensitive Cl(-) efflux has been identified as an important secretory pathway, the counterabsorptive pathways have not been identified. In other epithelial cells, the epithelial Na(+) channel (ENaC) has been identified as an important contributor to fluid absorption; however, its expression and function in BECs have not been previously studied. Our studies revealed the presence of α, ß, and γ ENaC subunits in human BECs and α and γ subunits in mouse BECs. In studies of confluent mouse BEC monolayers, the ENaC contributes to the volume of surface fluid at the apical membrane during constitutive conditions. Further, functional studies using whole-cell patch clamp of single BECs demonstrated small constitutive Na(+) currents, which increased significantly in response to fluid-flow or shear. The magnitude of Na(+) currents was proportional to the shear force, displayed inward rectification and a reversal potential of +40 mV (ENa+ = +60 mV), and were abolished with removal of extracellular Na(+) (N-methyl-d-glucamine) or in the presence of amiloride. Transfection with ENaCα small interfering RNA significantly inhibited flow-stimulated Na(+) currents, while overexpression of the α subunit significantly increased currents. ENaC-mediated currents were positively regulated by proteases and negatively regulated by extracellular adenosine triphosphate. CONCLUSION: These studies represent the initial characterization of mechanosensitive Na(+) currents activated by flow in biliary epithelium; understanding the role of mechanosensitive transport pathways may provide strategies to modulate the volume and composition of bile during cholestatic conditions. (Hepatology 2016;63:538-549).

The discovery of mesoporous molecular sieves from the twenty year perspective.

By the combination of prior knowledge, observation skills, and novel synthetic approaches, we discovered a family of mesoporous molecular sieves including discrete structures - MCM-41 (hexagonal), MCM-48 (cubic), and MCM-50 (lamellar). These materials were formed unlike that of our classical microporous structures involving reagent induced-macromolecular templating mechanism. Based on synthetic data and working with others, we were able to establish a predictive mechanism of formation and identify a broad class of templating reagents. These initial findings generated great interest and effort worldwide. It resulted in tremendous expansion of knowledge and skills with many new additional discoveries that established a new area of ordered mesoporous materials. They are integrated with zeolites (microporous materials) and based on surfactant inorganic chemistry.

Mechanosensitive Cl- secretion in biliary epithelium mediated through TMEM16A.

Bile formation by the liver is initiated by canalicular transport at the hepatocyte membrane, leading to an increase in ductular bile flow. Thus, bile duct epithelial cells (cholangiocytes), which contribute to the volume and dilution of bile through regulated Cl(-) transport, are exposed to changes in flow and shear force at the apical membrane. The aim of the present study was to determine if fluid flow, or shear stress, is a signal regulating cholangiocyte transport. The results demonstrate that, in human and mouse biliary cells, fluid flow, or shear, increases Cl(-) currents and identify TMEM16A, a Ca(2+)-activated Cl(-) channel, as the operative channel. Furthermore, activation of TMEM16A by flow is dependent on PKCα through a process involving extracellular ATP, binding purinergic P2 receptors, and increases in intracellular Ca(2+) concentration. These studies represent the initial characterization of mechanosensitive Cl(-) currents mediated by TMEM16A. Identification of this novel mechanosensitive secretory pathway provides new insight into bile formation and suggests new therapeutic targets to enhance bile formation in the treatment of cholestatic liver disorders.

Regulation of purinergic signaling in biliary epithelial cells by exocytosis of SLC17A9-dependent ATP-enriched vesicles.

ATP in bile is a potent secretogogue, stimulating biliary epithelial cell (BEC) secretion through binding apical purinergic receptors. In response to mechanosensitive stimuli, BECs release ATP into bile, although the cellular basis of ATP release is unknown. The aims of this study in human and mouse BECs were to determine whether ATP release occurs via exocytosis of ATP-enriched vesicles and to elucidate the potential role of the vesicular nucleotide transporter SLC17A9 in purinergic signaling. Dynamic, multiscale, live cell imaging (confocal and total internal reflection fluorescence microscopy and a luminescence detection system with a high sensitivity charge-coupled device camera) was utilized to detect vesicular ATP release from cell populations, single cells, and the submembrane space of a single cell. In response to increases in cell volume, BECs release ATP, which was dependent on intact microtubules and vesicular trafficking pathways. ATP release occurred as stochastic point source bursts of luminescence consistent with exocytic events. Parallel studies identified ATP-enriched vesicles ranging in size from 0.4 to 1 µm that underwent fusion and release in response to increases in cell volume in a protein kinase C-dependent manner. Present in all models, SLC17A9 contributed to ATP vesicle formation and regulated ATP release. The findings are consistent with the existence of an SLC17A9-dependent ATP-enriched vesicular pool in biliary epithelium that undergoes regulated exocytosis to initiate purinergic signaling.

Identification and functional characterization of TMEM16A, a Ca2+-activated Cl- channel activated by extracellular nucleotides, in biliary epithelium.

Cl(-) channels in the apical membrane of biliary epithelial cells (BECs) provide the driving force for ductular bile formation. Although a cystic fibrosis transmembrane conductance regulator has been identified in BECs and contributes to secretion via secretin binding basolateral receptors and increasing [cAMP](i), an alternate Cl(-) secretory pathway has been identified that is activated via nucleotides (ATP, UTP) binding apical P2 receptors and increasing [Ca(2+)](i). The molecular identity of this Ca(2+)-activated Cl(-) channel is unknown. The present studies in human, mouse, and rat BECs provide evidence that TMEM16A is the operative channel and contributes to Ca(2+)-activated Cl(-) secretion in response to extracellular nucleotides. Furthermore, Cl(-) currents measured from BECs isolated from distinct areas of intrahepatic bile ducts revealed important functional differences. Large BECs, but not small BECs, exhibit cAMP-stimulated Cl(-) currents. However, both large and small BECs express TMEM16A and exhibit Ca(2+)-activated Cl(-) efflux in response to extracellular nucleotides. Incubation of polarized BEC monolayers with IL-4 increased TMEM16A protein expression, membrane localization, and transepithelial secretion (I(sc)). These studies represent the first molecular identification of an alternate, noncystic fibrosis transmembrane conductance regulator, Cl(-) channel in BECs and suggest that TMEM16A may be a potential target to modulate bile formation in the treatment of cholestatic liver disorders.

Adenosine triphosphate release and purinergic (P2) receptor-mediated secretion in small and large mouse cholangiocytes.

UNLABELLED: Adenosine triphosphate (ATP) is released from cholangiocytes into bile and is a potent secretogogue by increasing intracellular Ca²(+) and stimulating fluid and electrolyte secretion via binding purinergic (P2) receptors on the apical membrane. Although morphological differences exist between small and large cholangiocytes (lining small and large bile ducts, respectively), the role of P2 signaling has not been previously evaluated along the intrahepatic biliary epithelium. The aim of these studies therefore was to characterize ATP release and P2-signaling pathways in small (MSC) and large (MLC) mouse cholangiocytes. The findings reveal that both MSCs and MLCs express P2 receptors, including P2X4 and P2Y2. Exposure to extracellular nucleotides (ATP, uridine triphosphate, or 2',3'-O-[4-benzoyl-benzoyl]-ATP) caused a rapid increase in intracellular Ca²(+) concentration and in transepithelial secretion (I(sc)) in both cell types, which was inhibited by the Cl(-) channel blockers 5-nitro-2-(-3-phenylpropylamino)-benzoic acid (NPPB) or niflumic acid. In response to mechanical stimulation (flow/shear or cell swelling secondary to hypotonic exposure), both MSCs and MLCs exhibited a significant increase in the rate of exocytosis, which was paralleled by an increase in ATP release. Mechanosensitive ATP release was two-fold greater in MSCs compared to MLCs. ATP release was significantly inhibited by disruption of vesicular trafficking by monensin in both cell types. CONCLUSION: These findings suggest the existence of a P2 signaling axis along intrahepatic biliary ducts with the "upstream" MSCs releasing ATP, which can serve as a paracrine signaling molecule to "downstream" MLCs stimulating Ca²(+)-dependent secretion. Additionally, in MSCs, which do not express the cystic fibrosis transmembrane conductance regulator, Ca²(+)-activated Cl(-) efflux in response to extracellular nucleotides represents the first secretory pathway clearly identified in these cholangiocytes derived from the small intrahepatic ducts.

Initiation of purinergic signaling by exocytosis of ATP-containing vesicles in liver epithelium.

Extracellular ATP represents an important autocrine/paracrine signaling molecule within the liver. The mechanisms responsible for ATP release are unknown, and alternative pathways have been proposed, including either conductive ATP movement through channels or exocytosis of ATP-enriched vesicles, although direct evidence from liver cells has been lacking. Utilizing dynamic imaging modalities (confocal and total internal reflection fluorescence microscopy and luminescence detection utilizing a high sensitivity CCD camera) at different scales, including confluent cell populations, single cells, and the intracellular submembrane space, we have demonstrated in a model liver cell line that (i) ATP release is not uniform but reflects point source release by a defined subset of cells; (ii) ATP within cells is localized to discrete zones of high intensity that are approximately 1 mum in diameter, suggesting a vesicular localization; (iii) these vesicles originate from a bafilomycin A(1)-sensitive pool, are depleted by hypotonic exposure, and are not rapidly replenished from recycling of endocytic vesicles; and (iv) exocytosis of vesicles in response to cell volume changes depends upon a complex series of signaling events that requires intact microtubules as well as phosphoinositide 3-kinase and protein kinase C. Collectively, these findings are most consistent with an essential role for exocytosis in regulated release of ATP and initiation of purinergic signaling in liver cells.

Identification and functional characterization of the intermediate-conductance Ca(2+)-activated K(+) channel (IK-1) in biliary epithelium.

In the liver, adenosine triphosphate (ATP) is an extracellular signaling molecule that is released into bile and stimulates a biliary epithelial cell secretory response via engagement of apical P2 receptors. The molecular identities of the ion channels involved in ATP-mediated secretory responses have not been fully identified. Intermediate-conductance Ca(2+)-activated K(+) channels (IK) have been identified in biliary epithelium, but functional data are lacking. The aim of these studies therefore was to determine the location, function, and regulation of IK channels in biliary epithelial cells and to determine their potential contribution to ATP-stimulated secretion. Expression of IK-1 mRNA was found in both human Mz-Cha-1 biliary cells and polarized normal rat cholangiocyte (NRC) monolayers, and immunostaining revealed membrane localization with a predominant basolateral signal. In single Mz-Cha-1 cells, exposure to ATP activated K(+) currents, increasing current density from 1.6 +/- 0.1 to 7.6 +/- 0.8 pA/pF. Currents were dependent on intracellular Ca(2+) and sensitive to clotrimazole and TRAM-34 (specific IK channel inhibitors). Single-channel recording demonstrated that clotrimazole-sensitive K(+) currents had a unitary conductance of 46.2 +/- 1.5 pS, consistent with IK channels. In separate studies, 1-EBIO (an IK activator) stimulated K(+) currents in single cells that were inhibited by clotrimazole. In polarized NRC monolayers, ATP significantly increased transepithelial secretion which was inhibited by clotrimazole. Lastly, ATP-stimulated K(+) currents were inhibited by the P2Y receptor antagonist suramin and by the inositol 1,4,5-triphosphate (IP3) receptor inhibitor 2-APB. Together these studies demonstrate that IK channels are present in biliary epithelial cells and contribute to ATP-stimulated secretion through a P2Y-IP3 receptor pathway.

Fluid flow induces mechanosensitive ATP release, calcium signalling and Cl- transport in biliary epithelial cells through a PKCzeta-dependent pathway.

ATP in bile is a potent secretogogue, stimulating cholangiocyte Cl- and fluid secretion via binding to membrane P2 receptors, though the physiological stimuli involved in biliary ATP release are unknown. The goal of the present studies was to determine the potential role of fluid flow in biliary ATP release and secretion. In both human Mz-Cha-1 biliary cells and normal rat cholangiocyte monolayers, exposure to flow increased relative ATP release which was proportional to the shear stress. In parallel studies, shear was associated with an increase in [Ca2+]i and membrane Cl- permeability, which were both dependent on extracellular ATP and P2 receptor stimulation. Flow-stimulated ATP release was dependent on [Ca2+]i, exhibited desensitization with repetitive stimulation, and was regulated by PKCzeta. In conclusion, both human and rat biliary cells exhibit flow-stimulated, PKCzeta-dependent, ATP release, increases in [Ca2+]i and Cl- secretion. The finding that fluid flow can regulate membrane transport suggests that mechanosensitive ATP release may be a key regulator of biliary secretion and an important target to modulate bile flow in the treatment of cholestatic liver diseases.